Smooth muscle and nonmuscle myosin II are regulated by phosphorylation of the 20 kDa regulatory light chain (RLC) located in the neck region. This region contains a single alpha-helical segment of the myosin heavy chain and the RLC and the essential light chain (ELC) and is called the regulatory domain. Previous studies have noted that single-headed species such as S-1 and single-headed myosin prepared by proteolysis of smooth muscle myosin have a high MgATPase activity regardless of the state of RLC phosphorylation. However, both of the species have a single regulatory domain. To determine whether interactions between adjacent regulatory domains of the two heads of myosin are essential for regulation, we have made a single-headed HMM-like molecule containing two regulatory domains using baculoviral expression of nonmuscle myosin IIB fragments. Sf9 cells were simultaneously infected with three viruses. One encoded an HMM-length fragment containing residues 1-1263. The second encoded residues 816-1263 tagged at the C-terminus with the FLAG epitope. This fragment binds both the RLC and ELC and forms a coiled-coil dimer. The third virus expressed both the RLC and ELC. A combination of FLAG affinity column and ATP-dependent binding to actin was used to purify the heterodimeric single-headed molecule. Nondenaturing gels and rotary shadowing EM confirmed that a single-headed fragment had been prepared. Its steady-state actin-activated MgATPase was activated 3-7-fold by RLC phosphorylation. Since steady-state measurements typically underestimate the degree of myosin's regulation, we are currently using single-turnover kinetic experiments to determine the rate constant for product release in the off state. These studies reveal that none of the single-headed framgents are as well regulated as the double-headed fragment, but that the turnover rate of a single-headed frament containing an intact regulatory domain was still activated 10-fold by phosphorylation. This suggests that the single-headed preparation has heterogenous kinetics and has both a fast and a slow phase to its ATPase activity. We are currently using stopped-flow fluorimetry to measure the rate of the fast phase and the amplitudes of both phases. Previous work in our lab has revealed that nonmuscle myosin IIA and myosin IIB have differing kinetic and motile properties. In a separate line of studies, we are engineering myosin IIA and myosin IIB S-1-like fragments for baculoviral expression. These fragments should allow us to determine the kinetic rate and equilibrium constants for various intermediate steps involved in the hydrolysis of MgATP.